A novel tubulin depolymerizing factor found recently can cause vascular occlusion at the dose lower than MTD (Expert Opin lnvestig Drugs.2004 September; 13(9) 1171-82). In 2005, Loin Vincent et al. came up with a novel tubulin depolymerizing factor with similar attributes that can, as vascular target agents (VTAs), damage tubulin skeleton, and documental data shows that the vascular target agents can selectively induce deterioration of tumor vessels, partially through VE-cadherin signal paths. Such a tubulin depolymerizing factor causes selective damage to tumor vessels and prevents angiogenesis of tumor without having an influence on normal vascular system. Meanwhile, it can inhibit aggregation of tubulin, selectively cause dysfunction and structural damage of tumor vessels and induce apoptosis of vascular endothelial cells in order to play the role of killing tumor cells or inhibiting tumor metastasis in case that the tumor cells are free from the support of nutrition and oxygen.
In 2005, GillianM.Tozer et al. reported in the influential magazine Nature Rev Cancer that: such compounds have an influence not only on the proliferation of vascular endothelial cells, but also on the migration of endothelial cells to further rapidly change the morphology of vascular endothelial cells, lead to the apoptosis of endothelial cells and break off the connection of vascular endothelial cells, thereby rapidly causing dysfunction and structural damage of tumor vessels. Since normal vessels are all supported by smooth muscle cells generally, such compounds that only act on the vessels without the support of smooth muscle cells have no influence on smooth muscle-supported vessels, in order to rapidly and selectively cause dysfunction and structural damage of tumor vessels to further selectively act on tumor cells and greatly reduce toxicity to normal cells (Nat rve Canaer.2005 June; 5 (6) 423-35 J. Clin. Invest. , Novenber 1, 2005; 115(11), 2992-3006). Such drugs are considered to be one of the most promising antitumor drugs at present.
Currently, among all the domestic and overseas researches on such drugs, only CombretastatinA-4 diphenylethylene compounds are put into clinical research, the patent of invention entitled ‘ethoxycombretastatin and preparation and uses of prodrug thereof’ (International Publication Number WO2008/031333A1) discloses that diphenylethylene B aromatic ring position 4′ alkoxy of CombretastatinA-4 is an active action site, original methyl at diphenylethylene B aromatic ring position 4′ is modified to ethyl that can form an active target with hydroxy, amino and other groups at position 3′, thus the target activity thereof for tumor vessels can be enhanced. However, owing to double-bond connection, family units in cis-configuration in Combretastatin causes the most effective damage to tumor vessels while family compounds in trans-configuration have no inhibiting effect on tumors. Cis-trans isomerization reaction exists and trans-configuration brings no drug effect but certain toxic and side effects, so the requirements of separation and purification technologies are high, the column chromatography is required, the consumption of now materials is large, the technological cost is high, the yield is low, and simultaneously, diphenylethylene compounds are transformed to trans-configuration through ultraviolet illumination and are required to be stored away from sunlight at low temperature, hence, the storage and actual application of the diphenylethylene compounds are extremely difficult.
The patent (Cushman,Mark et al. Synthesis and evaluation of analogs of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethane as potential cytotoxic andantimitotic agents, Journal of Medicinal Chemistry, 1992, Vol. 35, No. 12, 2293-306) discloses a compound, i.e. (Z)-1-(3,4,5-trimethoxy)phenyl-2-(4′-ethoxy)phenylethylene, however, synergistic active target cannot be formed as substituted groups such as —OH, —NH2 are not present at position 3′, and the anticancer drug effect gradually decreases from 4′-methoxy, ethoxy, propoxy at position 4′; and U.S. Pat. No. 6,054,598 discloses a synthetic method for modifying 2-methoxyestradiol to 2-ethoxyestradiol, 2-ethoxyestradiol includes the in vitro anticancer activity 1000 times as much as 2-methoxyestradiol; and researches have found that: ethoxydiphenylethane derivatives, 4′-ethoxy and 3′-hydroxy, amino have the same synergistic effect and can obviously enhance anticancer effect, but the anticancer effect thereof is remarkably lowered subsequent to the modification at position 4′ by propoxy.
Invention Contents:
1. The invention provides an ethoxydiphenylethane derivative, having the structure shown as the formula (I):

Wherein, the R is hydroxy, amino, phosphate, sulfate, choline phosphate, or amino acid side chain and water soluble ammonium salt thereof.
The R preferably is hydroxy, amino, disodium phosphate salt, ammonium phosphate salt, sulfate salt, choline phosphate inner salt, natural amino acid side chain and water soluble ammonium salt thereof, or —NH(COCHR′NH)m-H (wherein R′ is hydrogen, phenyl, and m represents an integer from 1 to 3) and water soluble ammonium salt thereof.
In preferred embodiments, the R is —OH, —NH2, —OPO2Na2, —NHCOCH2NH2 or —NHCOCHNH2CH2OH.
2. The invention discloses a preparation method of hydroxyethoxydiphenylethane derivative in the compound of the formula (I), which comprises the steps that:
(1) Under phase transfer catalysis, 4-hydroxy-3-methoxybenzaidehyde is subjected to ethoxylation by bromethyl to form 4-ethoxy-3-methoxybenzaldehyde III;

(2) meta-methyl is selectively removed by lithium diphenylphosphide and converted to hydroxy in order to obtain 4-ethoxy-3-hydroxybenzaldehyde IV;

(3) The 4-ethoxy-3-hydroxybenzaldehyde IV is prepared to 4-ethoxy-3-benzyloxybenzaldehyde V by benzyl chloride;

(4) 3,4,5-trimethoxy triphenyl benzylidene bromide phosphonium bromide tetrahydrofuran solution and 4-ethoxy-3-benzyloxybenzaldehyde are subjected to vinylation addition under the addition of potassium tert-butoxide in order to synthesize 3,4,5-trimethoxy-3′-benzyloxy-4′-ethoxydiphenylethylene VI;

(5) the 3,4,5-trimethoxy-3′-benzyloxy-4′-ethoxydiphenylethylene (VI) is hydrogenated under palladium-carbon to hydrogenate olefinic bonds, and debenzylation is performed to obtain 3,4,5-trimethoxy-3′-hydroxy-4′-ethoxydiphenylethane VII (hereinafter referred to as the code: ECB1);

(6) the 3,4,5-trimethoxy-3′-hydoxy-4′-ethoxydiphenylethane (VII) is subjected to phosphorylation, phosphate esterification and sulfation to form ethoxyhydroxydiphenylethane water soluble derivative: disodium phosphate salt, sulfate salt, ammonium phosphate salt or choline phosphate inner salt.
(7) ECB1 forms 3,4,5-trimethoxy-4′-ethoxydiphenylethane-3′-o-disodium phosphate salt VII (hereinafter referred to as the code: ECB1P) under the action of phosphorylation agent phosphorus oxychloride and 2 mol/L of NaOH.

(8) another preferred phosphate esterification is characterized in that: ECB1 is reacted with dibenzyl phosphate to form benzyl phosphate, and sodium methoxide/absolute methanol is added under trimethylbromosilane (TMBS) to obtain 3,4,5-trimethoxy-4′-ethoxydiphenylethane-3′-o-disodium phosphate salt (hereinafter referred to as the code: ECB1P).
Another preferred embodiment of the invention provides a preparation method of aminoethoxydiphenylethylene in the compound of the formula (I), which comprises the steps that:
(1) Under phase transfer catalysis, 4-hydroxy-3-nitrobenzaldehyde IX is subjected to ethoxylation by bromethyl to form 4-ethoxy-3-nitrobenzaldehyde X;

(2) trimethoxyphenyl bromide triphenylphosphonium methylide and the 4-ethoxy-3-nitrobenzaldehyde X are subjected to Wittig reaction to generate 3,4,5-trimethoxy-3′-nitryl-4′-ethoxydiphenylethylene XI;

(3) the 3,4,5-trimethoxy-3′-nitryl-4′-ethoxydiphenylethylene XI is subjected to hydrogenation reduction under palladium-carbon catalyst/sodium borohydride to reduce nitryl to amino and reduce olefinic bonds to ethane single bonds, so as to obtain 3,4,5-trimethoxy-3′-amino-4′-ethoxydiphenylethane XII (hereinafter referred as to the code: ECB1N)

(4) the 3,4,5-trimethoxyl-3′-amino-4′-ethoxydiphenylethane (XII) and amino acid derivatives are subjected to reaction to form ethoxyaminodiphenylethane amino acid amide derivative having the amino acid amide side chain as below: natural amino acid side chain, or —NH(COCHR′NH)m-H (wherein R′ is hydrogen, phenyl, and m represents an integer from 1 to 3).
(5) under the catalysis of dicyclohexylcarbo-diimide (DCC) and 1-hydroxybenzotrizole (HOBt) or hexafluorophosphatebenzotrizole-1-yl-oxo-tri(dimethylamino)phosphor(BOP agent), the 3,4,5-trimethoxy-3′-amino-4′-ethoxydiphenylethane XII is reacted with N-a-9-fluorenylmethoxycarbonyl amino acid derivative (Fmoc AA), amino at position 3′ is converted into Fmoc-amino acid amide, Fmoc is removed to generate amino acid amides of ECB1N, which respectively are 3,4,5-trimethoxy-3′-glycylamino-4′-ethoxydiphenylethane XIII (hereinafter referred as to the code: ECB1GN) and 3,4,5-trimethoxy-3′-serylamino-4′-ethoxydiphenylethane XIV (hereinafter referred as to the code: ECB1SN)

(6) the amino acid amide derivatives are dissolved in methanol, ethanol or isopropanol, and the equivalent amount of hydrochloric acid, sulfuric acid or phosphoric acid as well as petroleum ether or n-hexane are added to dilute the derivatives to form water soluble ammonium salt.
3. The pharmaceutical preparation of the invention is selected from the group consisting of the following forms: lyophiled powder, powder, injection, liposome, emulsion, micro-capsule, suspension or solution, administered in the form of intravenous injection; granule, tablet, capsule or syrup, administered orally; or suppository.
4. The use of the compound of the formula (I) in preparing tubulin aggregation inhibitor is provided.
5. The use of the compound of the formula (I) in preparing medicines having, as anti-tumor angiolysis agent, vascular target effect for various tumors is provided. The various tumors consist mainly of: lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, gastric cancer, bone cancer, esophagus cancer, breast cancer, prostatic cancer, testicular cancer, colorectal cancer, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cystic adenoic carcinoma, cystocarcinoma, medullary cancer, bronchiolar carcinoma, bone cell carcinoma, epithelial carcinoma, cancer of biliary duct, choriocarcinoma, embryo carcinoma, spermatocytoma, embryonal adenomyosarcoma, spongiocytoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymocytoma, pinealoma, hemocytoblastoma, vocal cord neuroma, meningoma, neuroblastoma, opticneuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromyxoma, fibro-osteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondroma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, myxolioma, chondrosarcoma, chondroma, chondromyoma, notochordoma, chorioadenoma, chorionepithelioma, chorionic epithelioma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, fibroosteoma, fibrosarcoma of bone, angiosarcoma, hemangioma, angiolipoma, angiochondroma, angioblastoma, angiokeratoma, angioglioma, hemangiosarcoma, angiofibroma, angiomyoma, angiolipoma, hematolymphangioma, angiolipoleiomyoma, angiomyoliopma, angiomyoneuroma, angiomyxoma, angioreticuloendothelioma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphadenoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, endoblastoma, synovioma, synoviosarcoma, mesolepidoma, mesocytoma, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic disease cytosis and erythrocytosis, lymphoma and multiplemyeloma.
6. The use of the compound of the formula (I) in preparing medicines for the treatment of diseases caused by abnormal angiogenesis is provided. The diseases mainly consist of: rheumatic arthritis, diabetic retinopathy, retinopathy of prematurity, retinal vein obstruction, psoriasis, rosacea, Kaposi's sarcoma, specific reaction keratitis, epidemic keratocon junctivitis, neovascular glaucoma, bacterial ulcer, mycotic ulcer, simple herpesvirus infection, zoster herpesvirus infection, protozoal infection, mycobacterium infection, polyarteritis, sarcoid, sclerotitis, rubeosis, arthritis syndrome with symptoms of dry mouth and eyes, systemic lupus erythematosus, acquired immune deficiency syndrome and syphilis.
7. The drug effect, safety evaluation and positive controls of the compound of the formula (I) are as below:
(Z)-3,4,5-trimethoxy-3′-hydroxy-4′-methoxydiphenylethylene XV (hereinafter referred to as the code: CA4);
(Z)-3,4,5-trimethoxy-4′-methoxydiphenylethylene-3′-o-disodium phosphate salt XVI (hereinafter referred to as the code: CA4P);

(Z)-3,4,5-trimethoxy-3′-amino-4′-methoxydiphenylethylene XVII (hereinafter referred to as the code: CA4N);

3,4,5-trimethoxy-3′-hydroxy-4′-methoxydiphenylethane XVIII (hereinafter referred to as the code: CB1)
3,4,5-trimethoxy-4′-methoxydiphenylethane3′-o-disodium phosphate salt XIV (hereinafter referred to as the code: CB1P);

3,4,5-trimethoxy-3′-amino-4′-methoxydiphenylethane XX (hereinafter referred to as the code: CB1N)
3,4,5-trimethoxy-3′-glycylamino-4′-ethoxydiphenylethane XXI (hereinafter referred to as the code: CB1GN)

(Z)-3,4,5-trimethoxy-3′-hydroxy-4′-ethoxydiphenylethylene XXII (hereinafter referred to as the code: ECA4);
(Z)-3,4,5-trimethoxy-4′-ethoxydiphenylethylene-3′-o-disodium phosphate salt XXIII (hereinafter referred to as the code: ECA4P);

(Z)-3,4,5-trimethoxy-3′-amino-4′-ethoxydiphenylethylene XXIV (hereinafter referred to as the code: ECA4N);
(Z)-3,4,5-trimethoxy-3′-glycylamino-4′-ethoxydiphenylethane XXV (hereinafter referred to as the code: ECA4GN)

8. The drug effect and the safety evaluation results of the compound of the formula (I) are concluded as below:                (1) the result of the antitumor activity evaluation for in vitro cultured tumor cells shows that, by comparison, position 4′ ethoxydiphenylethane compounds ECB1 and ECB1N as well as position 4′ ethoxydiphenylethylene positive control compounds ECA4 and ECA4N have obvious and basically equivalent antitumor activities for multiple in vitro cultured tumor cells, the antitumor activities thereof are prominently stronger than those of position 4′ methoxy positive control compounds CA4, CB1 and CB1N (about 10 to 200 times), and as for colorectal cancer HT-29, ECB1N is about 200 times stronger than position 4′ methoxy positive control CB1 and ECB1 is about 100 times stronger than positive control CB1.        (2) the growth of solid tumors depends on vascular system, a part of tumor vascular endothelial cells under rapid proliferation depend more on microtubule to maintain intact structure owing to the shortage of intact myofilament structures, the rapid proliferation of proliferous human umbilical vein endothelial cells (HUVEC) depends more on microtubule to maintain intact structure, so the microtubule is usually used as in vitro model for tumor vascular endothelial cells and the human umbilical vein endothelial cells (HUVEC) are used as action objects in order to evaluate the anti-tumor vessel property of ethoxydiphenylethane derivatives, ethoxydiphenylethane derivative ECB1N with the IC50 of 6.8×10−4 μmol/L and ethoxydiphenylethane derivative ECB1 with the IC50 of 7.5×10−4 μmol/L have the proliferation inhibiting effect for human umbilical vein endothelial cells, so a tubulin aggregation inhibitor which is quite obviously stronger than position 4′ methoxy positive control compounds CA4, CB1 and CB1N (with the IC50 ranging from 4.8×10−3 to 7.7×10−3) is shown, indicating that the ethoxydiphenylethane derivative is a potential, quite strong tumor vascular target drug.        (3) the result of the tumor-inhibiting rate experiment of in vivo intravenous injection tested drugs to S180 sarcoma transplanted tumor in mice shows that, according to the administration proposal, all the tested compounds can obviously inhibit the growth of S180 sarcoma transplanted tumor in mice, and it is observable around the eighth day after drug administration that, by comparison, position 4′ ethoxydiphenylethane derivatives ECB1P. ECB1GN hydrochloride and ECB1SN hydrochloride as well as position 4′ ethoxydiphenylethylene positive control compounds ECA4P and ECA4GN hydrochloride both achieve the tendency of tumor shrinkage in drug administered group, reach over 60% of the tumor inhibiting rate in case of 50 mg/kg dose and have fundamentally equivalent therapeutic effects, which are obviously superior to the therapeutic effects, i.e. about 40% of the tumor inhibiting rate, of methoxy positive controls CB1GN hydrochloride, CB1P and CA4P in case of 100 mg/kg dose.        (4) in the acute toxicity test of single mice intraperitoneal injection administration, high-dose injection administration causes the death of mice 40 minutes and 1 hour later, obvious residual liquid is not found after dissection, indicating the fast absorption of the drugs, and the other mice mainly die 1 to 2 days after the administration, no death of mice is observed after the fifth day, no abnormality of the heart, lung, liver, spleen, kidney and other organs in dead mice is found through dissection, and the surviving mice suffer from diarrhea not severe, which indicates that the tested drugs mainly lead to acute toxic response without obvious delayed toxicity, therefore, the result of the test shows that ethoxydiphenylethane compounds ECB1P, ECB1GN hydrochloride and ECB1SN hydrochloride have the toxicity lower than ethoxydiphenylethylene positive control compounds ECA4P and ECA4GN hydrochloride in administration group.        
9. The research of the invention has found that ethoxydiphenylethane compounds, i.e. the compounds of the formula (I), can form active target with hydroxy and amino at position 3′ by modifying original methoxy at diphenylethane B aromatic ring position 4′ into ethoxy, and can greatly enhance the tumor vascular target activity thereof compared with methoxydiphenylethane compounds with original methoxy at B aromatic ring position 4′ and hydroxy and amino at position 3′; the experiment shows that position 4′ ethoxydiphenylethane compounds ECB1 and ECB1N both have obvious antitumor activity for multiple in vitro cultured tumor cells and are prominently stronger than position 4′ ethoxy positive control compounds CA4. CB1 and CB1N (10 to 200 times), and as for colorectal cancer HT-29. ECB1N is about 200 times stronger than positive control CB1, and ECB1 is about 100 times stronger than positive control CB1.
Two benzene rings are connected through single bonds so that the compound has the structure, conformation, bonding force and reversal effect different from diphenylethylene compound CombretastatinA-4, in addition, no cis-trans configuration difference exists, drug stability can be greatly raised while toxicity can be lowered, preparation technology can be better simplified, no column chromatography separation is required, technological yield is prominently enhanced, consumption of raw materials is considerably reduced, technological cost of unit synthesis is also tremendously lowered, drug stability is raised, storage away from light is not required so as to bring great convenience for storage and actual application, and unexpected effects are obtained.